Clonal analysis of fetal hematopoietic stem/progenitor cells reveals how post-transplantation capabilities are distributed

Summary It has been proposed that adult hematopoiesis is sustained by multipotent progenitors (MPPs) specified during embryogenesis. Adult-like hematopoietic stem cell (HSC) and MPP immunophenotypes are present in the fetus, but knowledge of their functional capacity is incomplete. We found that fetal MPP populations were functionally similar to adult cells, albeit with some differences in lymphoid output. Clonal assessment revealed that lineage biases arose from differences in patterns of single-/bi-lineage differentiation. Long-term (LT)- and short-term (ST)-HSC populations were distinguished from MPPs according to capacity for clonal multilineage differentiation. We discovered that a large cohort of long-term repopulating units (LT-RUs) resides within the ST-HSC population; a significant portion of these were labeled using Flt3-cre. This finding has two implications: (1) use of the CD150+ LT-HSC immunophenotype alone will significantly underestimate the size and diversity of the LT-RU pool and (2) LT-RUs in the ST-HSC population have the attributes required to persist into adulthood.

1    the cell sorter.Libraries were prepared using CELSeq2 (Hashimshony et al., 2016) with additional optimizations, as described in (Amann-Zalcenstein et al., 2020).Single cells were sorted into plates containing a primer/lysis mix (dNTPs, ERCC and SUPERase Inhibitor).Plates were centrifuged for 1 min at 1,200 x g and immediately frozen down at -80°C until further processing.

Stonehouse et al Supplementary information Supplementary Figures and Legends
Sequencing reads were aligned and mapped to the mm10 mouse genome and ERCC spike-in sequences using Rsubread (Liao et al., 2019).GRCm38.88_chrannotation was then added.The data was demultiplexed and reads overlapping genes were summarized into counts using scPipe v1.6.0 (Tian et al., 2018).All subsequent analyses were performed in R version 3.6.0.Seven cells were identified as outliers based on number of genes detected, total gene counts, ERCC percentage, mitochondrial gene percentage and ribosomal gene percentage, and were excluded leaving 368 cells for downstream analysis.Gender-related genes were removed to avoid potential gender biases in the analysis.Lowly expressed genes were also filtered out such that 15590 genes showing an average count greater than 1 in at least 10 cells were retained.Normalisation factors were computed by deconvolution using scran v1.12.1 (Lun et al., 2016) and subsequently used to calculate log-transformed normalised expression values.Gene-specific variance of the biological and technical components of the data were then estimated.Cell cycle phases were predicted using the cyclone function in scran.Generalized log-linear models were then fitted to the count data, incorporating an adjustment for cell cycle phase.edgeR's likelihood ratio test pipeline was applied to identify differentially expressed genes between the cell populations (McCarthy et al., 2012).The false discovery rate (FDR) was controlled below 5% using the Benjamini Hochberg method.Code used for analysis will be made available on request.

Lentiviral barcoding
A maximum of 3 x 10 4 purified E14.5 FL LSK cells were transduced with SPLINTR virus in 96-well plates via spin-infection (90 mins at 1250 x g) at a multiplicity of infection of 0.01 -0.02 (this provided 1 -2% mCHERRY+ cells) to limit multiple integrations of the barcode per cell.Cells were washed and then injected intravenously into recipient mice.After two weeks mCHERRY+ barcoded cells were retrieved from the spleens of recipient mice two-weeks post transplantation.
Spleens were stained with markers for the isolation of monocytes (TER119 -MAC1 + Ly6G -[low side-scatter]), neutrophils (TER119 -MAC1 + LY6G + ), erythroblasts (TER119 + CD44 + ), and B cells (TER119 -MAC1 -B220 + ) (Figures S4, S5).mCHERRY + cell populations were sorted and spun into pellets of no more than 5 x 10 5 cells.Pellets were resuspended in 40 μL Direct PCR Lysis Reagent (Cell) with 0.5 mg/mL Proteinase K. Cells underwent lysis at 55°C for 2 hrs, followed by 85°C for 30 mins and 95°C for 5 mins.Libraries were generated as outline in (Naik et al., 2013).Raw sequencing FASTQ files were processed using custom C++ code to count SPLINTR barcode reads in each sample, including exclusively barcodes that to a previously generated reference library.All subsequent analyses were performed using the statistical computing language R. Counts below a threshold of 100 reads were set to zero.
Note that the final number of barcodes present in a given population is a function of: (1) Biological variation: this includes the abundance of the population (which dictates the number of cells that could carry a barcode), and the range of burst capabilities of the engrafting clones (the biomass contribution).Because little difference in hematopoietic reconstitution between freshly isolation and short-term cultures (Figure S3), the recovery of a large/small number of barcodes (clones) likely represents a biological feature of the population investigated.(2) Quality control measures: as shown in Figure S2, strict filtering of barcode sequencing data is essential to ensure that barcodes analysed are legitimately clonal.Scripts used for barcode analysis will be provided on request.

Figure S1 .
Figure S1.Representative examples for the gating strategy for the analysis of GFP donorderived hematopoietic reconstitution in sub-lethally irradiated GFP-recipient mice two weeks after transplantation.Data shown in this example is from the analysis of reconstitution derived from E14.5 fetal liver iST-HSCs.(A) Investigation of GFP+ reconstitution of monocytes in recipient (GFP-) peripheral blood.(B) Investigation of GFP+ reconstitution of platelets and erythrocytes in recipient (GFP-) peripheral blood.(C) Investigation of GFP+ reconstitution of natural killer (NK) and B cells in recipient (GFP-) spleen.(D) Investigation of GFP+ reconstitution of T cells in recipient (GFP-) thymus.